Skip to main content
ARS Home » Midwest Area » West Lafayette, Indiana » Crop Production and Pest Control Research » Research » Publications at this Location » Publication #58896
Title: PURIFICATION, CDNA CLONING AND CHARACTERIZATION OF PROTEINASE B, AN ASPARAGINE-SPECIFIC ENDOPEPTIDASE FROM GERMINATING VETCH (VICIA SATIVA L.) SEEDS

Author
item BECKER, CLAUDIA - IPK,GATERSLEBEN GERMANY
item SHUTOV, ANDREI - KISHINEV MOLDOVA CI/S
item NONG, VAN HAI - INST. BIOTECHNOL.,HANOI
item SENYUK, VITALI - MOLDAVIAN STATE UNIV.
item JUNG, RUDOLF - PURDUE UNIVERSITY
item HORSTMANN, CHRISTIAN - IPK,GATERSLEBEN,GERMANY
item FISCHER, JURGEN - IPK,GATERSLEBEN,GERMANY
item Nielsen, Niels
item MUNTZ, KLAUS - IPK,GATERSLEBEN,GERMANY

Submitted to: European Journal of Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: Enzymes called proteases regulate many cellular developmental processes. A newly recognized group of proteases, the asparaginyl endopeptidases, are found in legume seeds. Asparaginyl endopeptidases are involved in both the formation of cellular storage structures of seeds called protein bodies and the digestion of proteins in these cellular structures during seed germination. It is important to know whether asparaginyl endopeptidases derived from the same gene or different genes are involved in the cellular processes catalyzed by these enzymes during seed devleopment and germination. The research described in this manuscript concerns the purification and characterization of one such asparaginyl endopeptidase from vetch seed. This particular asparaginyl endopeptidase is active during seed germination. A DNA coding for the enzyme was identified, which made it possible to compare the amino acid sequence of this asparaginyl endopeptidase with one for a similar enzyme from castor bean that is activ during formation of protein bodies. The derived amino sequences of the two enzymes were quite similar. The results described are an important first step toward resolving the question whether the same or different enzymes are active during seed development and germination.

Technical Abstract: Proteinase B, an aspiragine-specific endopeptidase, was purified from germinating vetch seeds by two chromatographic steps. The final preparation consists of two proteins with molecular masses of about 39 and 37 kDa. Activity staining revealed that both the enzymes hydrolyze gelatin. Synthetic substrates were used to confirm cleavage specificity of fthe proteinase B preparation. The octapeptide DTRNGVEE was digested most efficiently. When Gly was replaced by lie or Glu, cleavage took place with lower efficiency. Polyclonal antibodies raised against the purified enzymes displayed both proteins in cotyledon extracts of germinated vetch seeds. In addition, a strong cross reacting protein band was found in cotyledon extracts of developing seeds indicating the presence of a very similar enzyme during seed development. Degenerate oligonucleotide primers were designed from the N-terminal amino acid sequence of Proteinase B, and this permitted cDNA clones to be obtained by means of polymerase chain reactions. The cDNA clones had an open reading frame of 1479 bp, and encoded a precursor polypeptide of 493 amino acids. The precursor displayed 59% sequence identify to the cDNA derived amino acid sequence of a vacuolar processing enzyme from developing castor bean endosperm. Using this cDNA as a probe, mRNA encoding the enzyme first became detectable at day 3 of germination and reached a maximum at day 8. Southern blot analysis suggested there were at least two genes for proteinase B.